Tooth loss through decay or accident is a major health problem in the USA, and genetic oligodontia occurs in almost 1% of the population. Current therapies rely on surgical implants that suffer from lack of sensitivity, mechanical failur and restricted utility in cases of bone loss. A long term goal of dental bioengineering is to use a patient's own cells for in vitro regeneration of tooth buds that can develop into normally functioning teeth following implantation. Recent progress towards this goal includes successful regeneration of rodent tooth buds from dissociated embryonic dental cells, or embryonic dental epithelia combined with adult mesenchymal stem cells, and implantation of these reconstructs to produce tooth structures that are innervated and vascularized. However, the sizes and shapes of these reconstructed structures are variable, indicating a need to identify endogenous factors that control these properties and could be applied in bioengineering strategies. Mutations of Wnt/beta-catenin pathway components in human patients are associated with dental defects. In particular, mutations in WNT10A are present in more than 50% of congenital non-syndromic hypodontia cases, as well as in a subset of ectodermal dysplasia syndromes. Genetic loss of function experiments in mice reveal requirements for Wnt/beta-catenin signaling at early stages of tooth development, and for formation of both cusp and root structures. Conversely, forced activation of oral epithelial beta-catenin in mouse embryos activates a cascade of downstream dental regulators, stimulating continuous tooth development, and in adults causes formation of embryonic-like tooth buds from incisor epithelia. The structures resulting from embryonic or adult activation of oral epithelial beta-catenin are, respectively, unicuspid, or ca mineralize but do not erupt. Thus, Wnt signaling must be tightly controlled for normal tooth morphogenesis. Wnt/beta-catenin activity is modulated by secreted antagonists, and by competition with alternate, beta-catenin independent pathways that are activated by specific combinations of Wnt ligands, receptors and co-factors. Based on published data and our preliminary studies we hypothesize that Wnt10a and its relative Wnt10b promote tooth development and molar cusp formation by activating beta-catenin signaling, and compete with secreted Sostdc1, Wif1 and Dkk family inhibitors to pattern tooth and cusp development. We further hypothesize that the Wnt receptor Fzd2 mediates non- canonical signaling by Wnt5a, which antagonizes the beta-catenin pathway and plays a critical role in tooth growth and cusp formation. We will test these hypotheses using genetic loss of function and rescue experiments and in vitro approaches. We will further utilize spatially and temporally-controlled delivery systems to determine whether recombinant WNT10A and Wnt10B proteins can be used to promote, and SOSTDC1, WIF1 and DKK proteins to inhibit, growth and cusp formation in embryonic tooth germs and tooth reconstructs in 3-D culture. Results from these experiments will suggest potential strategies for bioengineering teeth of defined size and shape, and for treating inherited conditions that affect tooth development.